The Study On The Rapid Detection Of Genetically Modiifed Plant Lines Using Loop-Mediated Isothermal Aplification

At present, transgenic technology got rapid development. According to the statistics fromISAAA, the global area of GM crops has consistently increased, reaching170.3millionhectares by2012, and which was approximately100-fold more than that in1996. So manycountries and regions have implemented the issues and regulations for the labeling andtraceability of GMF because of the people’s concern regarding the safety of GMF. To providethe technical support for the implementation of management and labeling regulations, rapid,sensitive, accurate and high-throughput methods for detection of GMF are urgent andimportant.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplificationmethod which was dveloped by Japanese researchers. In this study, real-time fluorescenceLAMP was established by using ESE tube scanner to collect SYBR Green I fluorescent signalto detect transgenic plant lines. The main research contents and results were as follows:(1) The specific LAMP primers of transgenic rice Bt63were designed according to thejunction sequences between the host genome and the inserted sequences. The reactionconditions were optimized to set up an appropriate real-time turbitidy LAMP. At the sametime, SYBR Green I concentration and FAM channel excitation intensity were optimized toset up an appropriate real-time fluorescence LAMP. The sensitivity, specificity and stability ofthese two methods were evaluated. The results showed that both methods were sensitive(0.01%), specific and stable. However, the detection time of real-time fluorescence LAMPwas shorter than real-time turbitidy LAMP, and ESE tube scanner was lighter and cheaperthan turbiditimeter. It was more suitable to use for field testing and recommend to grass-rootsunits.(2) The specific LAMP primers of transgenic soybean A5547-127, transgenic maizeMON89034and transgenic maize MIR604were designed and the real-time fluorescenceLAMP of these three lines were set up. The sensitivity, specificity and stability were evaluated.The results showed that they were sensitive (0.05%,0.1%and0.05%, respectively), specificand stable.(3) The detection capabilities of these four real-time fluorescence LAMP methods wereevaluated by testing foods and feed. The results were compared with the EU testing standardsby performing statistical analysis. The results showed there was no difference between real-time fluorescence LAMP and real-time PCR for the detection of A5547-127and MIR604. Thedetection rates of real-time fluorescence LAMP of Bt63and MON89034were slightly lower than real-time PCR, but LAMP technology had its own advantages. It was suitable for fieldtesting and recommending to grass-roots units.In summary, real-time fluorescence LAMP methods established in this study could meetthe testing standard of EU. They could provide technical support for the development of rapiddetection of GMF.